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The Effects of Sowing Date and Cultivars on Yield and Quality of Pea (Pisum sativum L.)

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Feride Öncan Sümer^*

This version is not peer-reviewed

Abstract

Peas are among the most widely consumed legumes and are of great benefit for human nutrition. Sowing and cultivation techniques are known to affect the yield and the quality of the seeds. Existing studies investigating the nutritional properties of the seeds from plants cultivated using different methods are continue to be of significant value. However, changes in the climate in recent years are presumed to have affected the optimum sowing dates for grain yield and quality. This study in-vestigates the effects of sowing peas at different sowing dates on the yield of the seed and on its nutrient content, as determined by certain quality traits (parameters). The experiment was con-ducted at the Aydin Adnan Menderes University Faculty of Agriculture in Turkey during the 2021-2022 and 2022-2023 pea production seasons. Specifically, it examined the yield of the seed (fresh/dry), its saponin and phenolic matter content and its amino acid composition in the case of five pea cultivars (Deren, Misya, Irmak, Karina, Local) sown at three different sowing times (November 15, November 30, December 15). The study found that the effects of sowing date on grain yield and quality characteristics were significant. However, the varieties' reactions to sowing dates were found to be different. The highest fresh seed yield (3.27 t ha-1) was obtained from samples sown at the second sowing date, while the highest dry yield (1.85 t ha-1) came from samples sown at the third sowing date. The cultivars also had a statistically significant effect on the yields. The highest fresh seed yields were obtained from the Misya (2.88 t ha-1) and Local (2.81 t ha-1) varieties, while the highest dry yields were obtained from samples of Local (1.76 t ha-1) and Irmak (1.73 t ha-1). In terms of sowing dates, the highest protein (27.75%) was obtained from the first sowing date, and the protein content decreased in the following dates. Misya was the cultivar from which the highest protein content (28.77%) was obtained. In the study, higher yields were obtained from the second and third sowing dates, while the first sowing time resulted in higher protein and amino acid composition. Similar to the protein content, the composition of almost all amino acids increased on the first sowing date. In addition, it was established that the saponin and phenolic substance contents of seeds varied with the sowing date.

Keywords:

Subject: Environmental and Earth Sciences - Other

1. Introduction

Pea is an important legume crop that provides a good source of protein, vitamins, minerals, and amino acids that are beneficial to human health. This legume is cultivated in almost all countries around the world and regarded as an essential part of the human diet. Pea is a particularly significant source of vegetable protein in vegetarian and vegan diets [1]. High protein content is essential for individuals who cannot consume meat and dairy products, helping them meet their protein needs [2].

Sowing date and variety selection are important management options to increase seed yield and protein content in such Mediterranean-type environments [3]. Many publications [4,5] have reported an increased yield with early sowing and a reduction in yield when sowing is delayed after the optimum time. These authors reported an advantage of early sowing dates, when combined with cultivars that avoid frost or cold damage at anthesis or in regions or seasons with low frost risk, aiming at high above-ground biomass at flowering to maximise radiation interception. Earlier studies have shown both the effect of climate on sowing and dates, crop development and variety choices, as well as other factors in addition to warming. Variety choices appear to vary by crop and region [6]. [7] suggested that sowing time may be even more critical on soils because that are prone to waterlogging in wheat. The sowing time may directly affect the yield of the plant through its influence on growth, flowering, pod formation and harvest [8]. Cool weather and mild climatic conditions provide optimum conditions for the pea, which grows more slowly in cold weather conditions. Choosing the right sowing time enables the plants to grow without winter damage, achieving healthy growth and productive yields.

Together with climatic conditions and other environmental factors, the time of sowing can also affect the phenolic compound content of plants [9]. These are natural compounds found in plants that have antioxidant properties [10] and also play an important role in the defense mechanisms of plants which protect them against environmental stresses [11]. Plants may produce different phenolic compounds at different stages of growth. At stages such as flowering and fruit formation, for example, their phenolic compound content may be particularly high. By choosing the right sowing date, the key stages of growth of the plant can be arranged accordingly and the levels of phenolic compounds at these stages can be increased. Some plants, such as peas, can activate defense mechanisms against environmental stresses by increasing their production of phenolic compounds under cold stress [12]. When the plants exposed to cold weather conditions, phenolic compounds are produced to neutralize the effect of an increase in free radicals. In this way, cells are protected against oxidative stress and the plant's resistance to cold increases [13].

Saponins are important antioxidant compound found in plants, serving as defense mechanisms and functioning to respond to environmental stresses. Factors such as temperature, humidity, and sunlight can affect the saponin content [14]. For example under stressful conditions (e.g. drought or pest attack), plants may produce more saponins as a defensive response. Many researchers have identified legumes as the main saponin-containing food in the human diet. Different varieties of pea may have different saponin profiles [15]. Therefore, the choice of sowing date may have different results in terms of saponin content even in the same variety of pea [16]. Choosing the most appropriate times of sowing and harvesting helps in obtaining the optimum saponin content [17].

Protein content is the primary determinant of quality in peas. Pea protein can be considered an advantageous alternative to soya protein since they are not genetically modified and have lower allergenicity [18]. In Mediterranean regions like Turkey, the planting period extends for more than 2 months. Sowing in the first half of November results in variability in flowering time. Late-sown varieties may experience heat stress during flowering, which can result in smaller seeds but high protein accumulation. Amino acids are the components of proteins. A study on lentil concluded that the time of harvest has an effect on seed chemistry, including protein, amino acid, and mineral content and it was indicate that sulfur-containing amino acids are typically the most deficient in legumes [19]. Little research has been conducted to investigate the impact of sowing time on the amino acid content in peas. Factors such as temperature, humidity and sunlight can also affect the amino acid content of the seeds [20]. For example, the amino acid synthesis and accumulation of plants may vary under cold weather conditions or during periods of drought [21]. Pea plants are harvested a specific period after they have been sowed. The time of harvesting also affects the amino acid content of the seed. Selecting the harvest time appropriately helps in obtaining the optimum amino acid content in the seed [22]. One study on legumes has found significant differences between species in terms of all essential amino acids. Cysteine and methionine are the most limiting amino acids in legume seeds [23].

In brief, conducting planting trials to determine the optimal sowing dates that maximize crop yields and quality is essential for meeting the nutritional requirements of expanding populations and enhancing the sustainability of agriculture. Selecting the appropriate sowing date can help reduce the adverse effects of early frost damage and protect plants from high temperatures that may occur during critical stages such as pod setting and grain filling periods. Moreover, changing climatic conditions may lead to changes in the optimal dates for sowing and harvesting. The aim of this study was to determine the most suitable sowing date for pea plants in the province of Aydin, Turkey, which has a prevailing Mediterranean climate.

2. Material and Methods

2.1. Plant Material

Five pea (Pisum sativum L.) varieties (cultivars) were planted. Three of these (Karina, Misya and Local) are widely grown in the Aegean region of Turkey. These cultivars were sourced from producers who have been planting peas in the province of Aydin over a long period of time. The other two varieties (Deren and Irmak) were developed and provided by the Eastern Mediterranean Agricultural Research Institute (Adana, Turkey) in 2020.

2.2. Experimental Design

The experiment was carried out in the experimental field ("27°51'0" E, 37°51'0" N", altitude 50 m) of the Department of Field Crops of the Faculty of Agriculture of Aydin Adnan Menderes University, Aydin, Turkey in the 2021/22 and 2022/23 seasons. The experiment was carried out in a randomized complete block (RCB) design with split plot arrangements, having three replications. Main plots were used for the three different sowing dates (November 15, November 30 and December 15) and sub-plots were assigned for each of the five varieties (Deren, Misya, Irmak, Karina and Local). In all, there were 45 parcels, each of them covering 10.8 m². The parcels were 6 m long and had six rows with a distance of 30 cm between the rows. A target of 85 plants per square meter was adopted. Before sowing, 40 kg ha^-1 of nitrogen, phosphorus and potassium was applied in the form of 15-15-15 fertilizer. In addition, nitrogen fertilizer (20 kg N ha^-1) was applied after sowing and prior to flowering. Weed control was carried out manually twice, during and after flowering. No irrigation was conducted since the total annual rainfall was 500-550 mm. No pesticide was used. Harvesting was carried out manually from the middle 4 rows. The most common sowing date in the region was observed to be the end of November, although there are no available studies in this connection. For this reason, it was decided to plant the peas at three different times with intervals of two weeks between them – namely, November 15, November 30 and December 15 (Table 1 and Figure 1).

As seen in Table 1, the soil is of a sandy loam type. Organic matter content was low (1.2%) and phosphorus content was high. The calcium and sodium contents of the soil were also found to be low.

Figure 1 displays the long-term average temperature and precipitation, alongside the average temperature and total precipitation for the experimental years. The average temperature in April, which coincides with the pod formation period, exceeded the long-term average temperature in the first year of the experiment. The amount of precipitation in both study years was quite low when compared to the average levels for previous years. Although there was no need for irrigation during the pea growing period, the seed traits examined in the experiment were found to have been affected by low precipitation.

2.3. Yield Measurement

Fresh seed yield (t ha^-1): The seed yield obtained from the area of 7.2 m² (four rows) allocated to each replicate was calculated in t ha^-1. Dry seed yield (t ha^-1): The seed yield obtained from the 7.2 m² area allocated to each replicate after drying was calculated in t ha^-1.

Cold tolerance scoring was done on a scale from 0 to 5, in which 0 indicates no dam-age and 5 indicates that all the plants were killed (Singh et al. 1989). In calculating the score, account was taken of the damage to the stem and leaves of the plant and of the ratio of this damage to the overall plot. The following scheme was applied: 0 = no visible leaf damage, 2 = tolerant, 3 = moderately tolerant, 4 = moderately susceptible, 5 = 100% leaf damage or dead.

2.4. Quality Characters

Seed protein content (%): The seed protein content was measured using the Near In-frared Reflected Spectroscopy (NIRS) method with the Bruker German Multi-Purpose An-alyzer at the Adnan Menderes University Agricultural Biotechnology and Food Safety Application and Research Center (ADÜ-TARBİYOMER) [24].

Saponin analysis: 2.50g of pea flour (ground dry pea grains) was weighed and mixed with 25mL of 50% (v/v) ethanol. The mixture was subjected to extraction in a water bath (Mikrotest MCS-30, Turkey) for 120 minutes at 50°C and with a shaking speed of 50rpm. After extraction, the samples were filtered with filter paper and the filtrate was placed in a 25 mL balloon jug and filled to the volume line with 50% ethanol. The extracts were diluted by a ratio of 1:20 (v/v) in accordance with the Beer-Lambert rule spectrophotometric reading range. Then the saponin analysis was performed. 3.5 mL of Lieberman-Buc hard reagent (concentrated sulfuric acid containing 16.7% acetic acid anhydride) was added to 1mL of extract and the samples were vortexed and kept in the dark at room temperature for 30 minutes. At the end of this period, the absorbance value of the samples in the quartz cuvette was read on a spectrophotometer (SOIF UV-5100, China) at the 528nm wavelength. The calibration curve (y = 0.0009x - 0.0079; R2 = 0.99) was calculated using different concentrations (50-1,000mg/L) of saponin standard (Sigma-Aldrich, USA) and the results were expressed in mg/g [25,26,27].

Total phenolic matter analysis: The dried and ground pea samples were mixed with a 50% ethanol solution (0.01% HCl) at a ratio of 1:10 (w/v) and subjected to extraction for 120 minutes at 50°C with a shaking speed of 50 rpm. After extraction, the samples were centrifuged for 10 minutes at 6,000rpm) and the supernatant was collected in a tube. For the total phenolic matter analysis, 30µL of extract and 150µL of Folin-Ciocalteu reagent were added to 2.37mL of deionized water and kept in the dark for 8 minutes. Then 450µL of saturated sodium carbonate was added to the mixture, which was kept in an oven for 30 minutes at 40°C, after which the absorbance value was read on a spectrophotometer at the 750nm wavelength [28]. The total phenolic content of the pea samples was expressed in "mg gallic acid equivalent (GAE)/100g".

Seed amino acid content (g/100g): Dry seeds were ground after harvesting and random samples were prepared from each plot. A total of seventeen amino acids were measured. The seed amino acid analyses were performed by means of high-performance liquid chromatography or high-pressure liquid chromatography (high-performance liquid chromatography or high-pressure liquid chromatography, HPLC) at the Research and Application Center of the Drug Development and Pharmacokinetics Laboratory at the Ege University.

2.5. Statistical Analysis

The univariate procedure of the SAS (1999) [29] statistical package program was used to assess the normality of the data. The analysis results indicated that the data for all the measured characteristics were normally distributed. Subsequently, the GLM procedure of the same software was used to conduct variance analyses and obtain least squares means for the investigated characteristics. When statistical significance (P<0.05) was detected for all characteristics, paired comparisons between means were conducted using the Duncan test. To visualize the distribution and statistical properties of the data obtained from the study, box plot graphics were created using the same statistical software package.

3. Results and Discussion

Table 2 indicates the sources of the variations in traits found in the study together with their levels of significance. The year of the experiment was a significant factor for all the traits. For this reason, the averages of the traits were separated by years. Yield (fresh and dry), saponin, phenolic, and protein content interaction significance, as well as the mean and standard errors, are presented in Table 2 (2022) and Table 3 (2023).

Sowing dates were found to be significant for fresh seed yield values. The average fresh seed yield ranged from 2.10 to 3.27 tons per hectare (t/ha) (Table 3). In terms of sowing time, the highest fresh seed fresh yield (3.27 t ha^-1) (Table 3) was obtained from the second sowing. The sowing dates were ranked 2>3>1 (Table 2 and Table 3) according to the resulting fresh seed yield values. Among the varieties, Misya (2.88 and 2.85 t ha^-1), Local (2.81 and 2.79 t ha^-1) and Deren (2.77 and 2.79 t ha^-1) exhibited the highest fresh seed yield in two years (Table 2 and Table 3). However, in another study, early sowing (November 15) generated higher values for seed yield and traits affecting yield than late sowing (December 15) [30]. The yield in second and third, where irrigation was limited, was found to be lower than in first sowing in pea [31]. Higher than usual temperatures in April (see Figure 1) led to the rapid maturation of peas especially in third sowing date. The absence of irrigation and inadequate rainfall during this period could result in a decrease in crop yield. Plants sown at third sowing date might more affected by this situation, resulting in lower fresh yields [32]. Further studies shown that cumulative solar radiation during the seed filling period was positively related to yield (p = 0.009), probably due to an increase in seed weight [33]. It is likely that the plants exposed to sunlight during the pod-filling period in April to May for the second sowing date had a positive impact on seed production.

The average dry grain yield ranged from 0.87 to 1.85 t ha^-1 (Table 2 and Table 3). The highest value in terms of sowing dates was obtained from the third sowing date (1.85 t ha^-1) (Table 3), and the dry grain yield decreased as sowing was delayed. With respect to dry seed yield values, the sowing dates ranked 3>2>1 (Table 2 and Table 3). In the late spring, the increased day length and warmth initiate the generative period. This may have led to high yield of at the third sowing date. However, in previous studies, it was determined that changing the sowing date from autumn to spring caused a decrease in yield, and late sowing was avoided [34]. Recommending late sowing may not be appropriate due to cold damage that may occur in March [35]. The order of dry grain yield by variety is as follows: Local > Karina > Irmak > Misya > Deren (Table 3). Misya and Deren had high water content in seed and they lose weight when dried, so they might be low dry yield. In addition, the high maximum temperatures observed in March in the second year of the experiment led to low seed yields (Figure 1). The minimum, optimum and maximum temperatures for the emergence, vegetation and generative periods have been put at 3°C, 28°C and 38°C respectively [36]. In previous studies was shown to be higher yield in the March compared to February, and the reason for this was to be lower risk of early frost or cold damage in peas [37].

The average saponin values ranged from 49.76 to 102.40 mg/g (Table 2 and Table 3). As shown in Figure 3, according to the average saponin appears to be the trait most sensitive to the time of sowing. Saponins have been present in the structure of many legumes [38]. Saponin content can be affected by factors such as the cultivar, the location, the type of soil and the sowing and harvesting dates [39]. While the average value of saponin was high on the third sowing date (102.40 and 102.35 mg g-1) (Table 2 and Table 3), it was found to be lower in the early sowing dates. Saponins are known as compounds found in plants that have a deterrent effect against pests [40,41]. The high temperature coinciding with the pod formation period on the third sowing date may have increased the saponin content of the seed. High temperature may have triggered the plant defense mechanism and saponin accumulated. Generally, excessive amounts of this substance may lead to bitter or toxic effects. Since saponins are bitter, pea varieties with lower saponin content are preferred in food production [42]. Figure 3 shows that saponin values increased from the first sowing date to the last one. The high temperatures observed in April and May (Table 1) during the grain filling period may have activated the plant's defense mechanism. When the collected data are evaluated by variety, they are ranked as Misya > Deren > Karina > Local > Irmak (Table 2 and Table 3). The saponin contents of the varieties are influenced by both genotype and environment, and this interaction may result in varying saponin levels across the varieties [43].

In the study, it was observed that the average phenolic compound content values varied between 33.19 and 127.30 mg (GAE)/100 g (Table 2 and Table 3). In terms of sowing dates, the highest value was obtained during the third sowing date (Figure 3). The phenolic content may have increased during the third planting period because the harvest coincided with the first weeks of May, and the plants were exposed to high temperature for a longer duration [44]. It has been observed that the choice of cultivar can affect phenolic compound and saponin content phenolic compounds have been recognized to act as antioxidants and found in high amounts in peas [44]. The properties of antioxidant plant phenolic compounds and their effects in preventing various oxidative stress diseases have been identified [45]. In the present study, the average total phenolic content was found to vary significantly by cultivar. The highest phenolic content was obtained from Misya with 98.72 mg GAE/100g (Table 2) and the lowest from Karina with 38.09 mg GAE/100g (Table 3). Misya was one of the varieties with the highest cold damage. This may have affected the phenolic content as a result of the variety's response to cold damage

The average protein values were measured in the study ranged between 24.06 and 28.77 (%) (Table 2 and Table 3). The effect of sowing date on grain protein content was found to be statistically significant. The highest protein content (27.75%) (Table 2) was obtained from the first sowing date. This was followed by the third (26.67%) and second sowing date (26.55%) (Table 2). In this study, as in previous studies, we observed an inverse relationship between seed yield and protein content [46]. The protein content increased as the seed fresh yield decreased. Both proteins contain up to 29% dry matter (DM), with lysine about 7% of the total proteins. and carbohydrates (starch) in suitable proportions, making up to 59% of dry matter (DM), along with fiber and vitamins [47]. Among the varieties, Misya (28.77%) (Table 2) had the highest protein content. The order is Misya>Deren>Karina>Irmak>Local (Table 2). Similarly, in previous studies, protein content in soybean was found to be higher in early sowing date [48]. The quantity and the fractions contained determine the protein quality of the proteins and thus the quality of the wheat and it was determined that the temperature during the grain filling period was effective on protein content in wheat [49]. In a study conducted in beans, it was determined that the amount of protein increased as the sowing date was postponed, but the protein yield decreased due to low yield [50].

Interaction significance, means, and standard errors for amino acids are presented in Table 4/5 (2022) and Table 6/7 (2023). The average quantity of glutamic acid in the overall amino acid composition (2.96 and 2.87 g/100 g), followed by arginine (2.79 and 2.85 g/100 g) (Table 4 and Table 6), cysteine (2.25 and 2.24 g/100 g) (Table 5 and Table 7). The effect of sowing date and cultivars on amino acids was found to be statistically significant (Table 4, Table 5, Table 6 and Table 7). Similar findings have previously been obtained from tepary beans [51]. These amino acids are followed by aspartic acid (1.90 and 1.88 g/100 g) (Table 4 and Table 6), and leucine (1.32 and 1.31 g/100 g) (Table 5 and Table 7). Accordingly, leucine is the essential amino acid with the highest amount. Misya (1.38 g/100g) and Irmak (1.32 g/100g) (Table 5) have high average leucine levels on the third sowing date. Many researchers found that glutamic acid content in seeds was highest followed by aspartic acid and the lowest contents of amino acids methionine in legumes [52].

The variety factor was found to be important in valine. Cultivars were listed as Karina (1.00 g/100g)> Local (0.98g/100g)>Irmak (0.95g/100g)>Deren (0.94g/100g)>Misya (0.93g/100g) (Table 7). The highest average was measured from the first sowing date (0.99g/100g) (Table 7). Sowing date and variety factor on methionine were found to be statistically significant. The amount of methionine was found to be higher in the first sowing date (0.17 g/100g) (Table 5). Among the varieties, the amount of methionine in Local (0.16g/100g) (Table 5) was higher than the others. It is known that legumes generally contain low levels of sulfur-containing amino acids such as methionine, but previous studies have found a higher (0.94-1.25 g/100g) methionine content in beans [53]. It was found that sowing date and cultivar factor were statistically significant on phenylalanine. Sowing dates were ranked as 1 (0.74g/100g)>2=3 (0.72g/100g) (Table 5). Varieties were ranked as Karina (0.78)>Local (0.74g/100g)>Deren (0.72g/100g)>Misya =Irmak (0.70g/100g) (Table 5). Both sowing date and varieties had a statistically significant effect on isoleucine. Among the sowing dates, the first sowing date resulted in the highest average isoleucine (0.81g/100g) (Table 5). It has the highest content of Local (0.83 g/100 g) among the varieties (Table 5). The effect of sowing date and varieties on lysine was statistically significant. Proteins from legumes that are low in Sulphur-containing amino acids can be used effectively in combination with most of the proteins from cereal grains which are deficient in lysine [53]. Sowing dates gave average values as 1(0.81g/100g)>3(0.80g/100g)>2(0.79g/100g). The varieties are listed as Local (0.83 g/100g)> > Misya (0.81 g/100g)>Irmak (0.80 g/100g)>Deren(0.78 g/100g)>Karina (0.77 g/100g) (Table 7). Previous studies have shown that our lysine results were similar and peas have the highest lysine content among legumes [54]. The effect of sowing date and cultivars were found statistically significant in Threonine. The average threonine varied between 0.85-1.04 g/100g (Table4&6). The highest amount of threonine (0.97 g/100g) (Table 4)was obtained from the first sowing date. Karina>Irmak>Misya>Local>Deren (Table 4) when evaluated according to the varieties. In previous studies, higher amino acid content was observed in winter plantings compared to summer plantings [55]. Sowing date change the environmental conditions for seed which may affect protein content and composition. [56] declared the change of seed protein content and the sum of essential amino acids under the growing season and sowing date. But they also found that concentrations of cys, met and phe tyr, his and trp accumulation in grain protein were not affected by the sowing date.

Histidine varies between 0.39-0.43 g/100g (Table 4 and Table 6). The highest mean values were measured from the first sowing date (0.43 g/100g), followed by the second (0.41 g/100g) and third (0.40 g/100g) (Table 6) sowing dates. The varieties were ranked as Local>Misya>Deren>Karina=Irmak (Table 6). Serine varied between 0.84-0.92 g/100 g (Table 4 and Table 6).

Accordingly, serine values were found to be higher for the first sowing time and lower for the second sowing time (Figure 4). Varieties were found statistically significant in terms of serine. The varieties were ranked as Karina>Misya>Deren>Irmak>Local (Table 6). It is observed that glycine values were significantly affected by sowing date and cultivars and the average values varied between 0.64-0.72 g/100 g (Table 4 and Table 6). The highest value was measured from the first sowing date. However, among the varieties, Karina and Misya (0.72 and 0.71 g/100 g) (Table 6) gave higher values.

Table 4. Least squares means and standard errors for investigated characteristics belonging to 2022.

Factors	N	ASP	GLU	SER	HIS	GLY	THR	ARG	ALA	TYR
Sowing Date	N	P=0.112	P=0.000	P=0.000	P=0.000	P=0.603	P=0.447	P=0.666	P=0.041	P=0.095
1(November 15^th)	15	1.90±0.014	3.01±0.020^a	0.88±0.006^a	0.42±0.004^a	0.66±0.011	0.97±0.018	2.77±0.041	1.29±0.020^ab	0.52±0.006
2(November 30^th)	15	1.92±0.014	2.88±0.020^b	0.84±0.006^b	0.40±0.004^b	0.67±0.011	0.95±0.018	2.80±0.041	1.35±0.020^a	0.53±0.006
3(December 15^th)	15	1.88±0.014	2.99±0.020^a	0.87±0.006^a	0.39±0.004^b	0.66±0.011	0.94±0.018	2.82±0.041	1.28±0.020^b	0.52±0.006
Cultivars		P=0.000	P=0.000	P=0.000	P=0.001	P=0.032	P=0.000	P=0.036	P=0.012	P=0.000
Deren	9	1.81±0.018^a	2.99±0.026^a	0.84±0.008^c	0.39±0.006^b	0.66±0.014^ab	0.89±0.023^c	2.83±0.052^b	1.33±0.026^a	0.49±0.008^d
Irmak	9	1.93±0.018^b	3.10±0.026^b	0.87±0.008^b	0.39±0.006^b	0.65±0.014^b	0.97±0.023^bc	2.79±0.052^bc	1.29±0.026^ab	0.54±0.008^b
Karina	9	1.97±0.018^b	2.84±0.026^c	0.85±0.008^bc	0.39±0.006^b	0.69±0.014^a	1.04±0.023^a	2.83±0.052^b	1.35±0.026^a	0.50±0.008^c
Misya	9	1.94±0.018^b	2.81±0.026^c	0.85±0.008^bc	0.41±0.006^ab	0.68±0.014^a	0.95±0.023^b	2.88±0.052^a	1.33±0.026^a	0.56±0.008^a
Local	9	1.85±0.018^a	3.07±0.026^b	0.91±0.008^a	0.42±0.006^a	0.64±0.014^c	0.92±0.023^c	2.65±0.052^c	1.22±0.026^b	0.52±0.008^bc
SD*C		**	**	**	**	**	**	**	**	**
Overall		1.90±0.008	2.96±0.012	0.86±0.004	0.40±0.003	0.66±0.006	0.95±0.01	2.79±0.023	1.3±0.012	0.52±0.004

SD*C: Interaction between sowing date x Cultivars **: P<0.01 ASP: Aspartic acid, GLU: Glutamic acid, SER: Serine, HIS: Histidine, GLY: Glycine, THR: Threonine, ARG: Arginine, ALA: Alanine, TYR: 226 Tyrosine. All amino acid values in g 100 g^-1.

Table 5. Least squares means and standard errors for investigated characteristics belonging to 2022.

Factors	N	CYS	VAL	MET	PHE	ILE	LEU	LYS	PRO
Sowing Date	N	P=0.875	P=0.105	P=0.001	P=0.346	P=0.382	P=0.000	P=0.272	P=0.26
1(November 15^th)	15	2.25±0.034	0.97±0.034	0.17±0.006^a	0.74±0.009	0.81±0.008	1.32±0.01^b	0.78±0.009	0.66±0.016
2(November 30^th)	15	2.24±0.034	0.87±0.034	0.15±0.006^b	0.72±0.009	0.79±0.008	1.27±0.01^c	0.76±0.009	0.63±0.016
3(December 15^th)	15	2.27±0.034	0.94±0.034	0.14±0.006^b	0.72±0.009	0.80±0.008	1.38±0.01^a	0.77±0.009	0.67±0.016
Cultivars		P=0.14	P=0.523	P=0.235	P=0.000	P=0.000	P=0.001	P=0.002	P=0.007
Deren	9	2.29±0.044^b	0.93±0.044	0.15±0.007	0.72±0.012^bc	0.81±0.010^b	1.31±0.012^b	0.75±0.011^bc	0.61±0.020^a
Irmak	9	2.26±0.044^b	0.94±0.044	0.14±0.007	0.70±0.012^c	0.80±0.010^bc	1.32±0.012^b	0.77±0.011^b	0.71±0.020^c
Karina	9	2.16±0.044^ab	0.97±0.044	0.15±0.007	0.78±0.012^a	0.80±0.010^bc	1.30±0.012^b	0.74±0.011^c	0.68±0.020^bc
Misya	9	2.24±0.044^b	0.92±0.044	0.14±0.007	0.70±0.012^c	0.75±0.010^c	1.38±0.012^a	0.78±0.011^b	0.63±0.020^ab
Local	9	2.32±0.044^a	0.86±0.044	0.16±0.007	0.74±0.012^b	0.83±0.010^a	1.31±0.012^b	0.80±0.011^a	0.63±0.020^ab
SD*C		**	ns	**	**	**	**	**	**
Overall		2.25±0.02	0.92±0.02	0.15±0.003	0.73±0.005	0.8±0.004	1.32±0.006	0.77±0.005	0.65±0.009

SD*C: Interaction between sowing date x Cultivars **: P<0.01 Tyrosine, CYS: Cysteine, VAL: Valine, MET: Methionine, PHE: Phenylalanine, Isoleucine, LYS: Lysine, LEU: Leucine, PRO: Proline. All amino acid values in g 100 g^-1.

Table 6. Least squares means and standard errors for investigated characteristics belonging to 2023.

Factors	N	ASP	GLU	SER	HIS	GLY	THR	ARG	ALA	TYR
Sowing Date	N	P=0.118	P=0.000	P=0.000	P=0.000	P=0.649	P=0.450	P=0.626	P=0.114	P=0.084
1(November 15^th)	15	1.88±0.014	2.93±0.020^a	0.89±0.006^a	0.43±0.004^a	0.69±0.011	0.93±0.018	2.82±0.041	1.24±0.021	0.45±0.006
2(November 30^th)	15	1.90±0.014	2.79±0.020^b	0.85±0.006^b	0.41±0.004^b	0.70±0.011	0.91±0.018	2.84±0.041	1.29±0.021	0.46±0.006
3(December 15^th)	15	1.86±0.014	2.90±0.020^a	0.88±0.006^a	0.40±0.004^b	0.69±0.011	0.90±0.018	2.88±0.041	1.23±0.021	0.44±0.006
Cultivars		P=0.000	P=0.000	P=0.000	P=0.001	P=0.028	P=0.000	P=0.035	P=0.082	P=0.000
Deren	9	1.79±0.018^b	2.90±0.026^b	0.85±0.008^c	0.41±0.006^bc	0.69±0.014^abc	0.85±0.023^b	2.89±0.053^ab	1.27±0.027^a	0.42±0.008^c
Irmak	9	1.92±0.018^a	3.01±0.026^a	0.88±0.008^b	0.40±0.006^c	0.68±0.014^ab	0.93±0.023^a	2.84±0.053^b	1.23±0.027^ab	0.46±0.008^b
Karina	9	1.95±0.018^a	2.75±0.026^c	0.86±0.008^bc	0.40±0.006^c	0.72±0.014^c	1.00±0.023^a	2.87±0.053^a	1.29±0.027^a	0.43±0.008^bc
Misya	9	1.92±0.018^a	2.72±0.026^c	0.86±0.008^bc	0.42±0.006^ab	0.71±0.014^bc	0.91±0.023^ab	2.93±0.053^a	1.28±0.027^a	0.49±0.008^a
Local	9	1.83±0.018^b	2.98±0.026^a	0.92±0.008^a	0.43±0.006^a	0.67±0.014^a	0.88±0.023^ab	2.70±0.053^b	1.19±0.027^b	0.45±0.008^ab
SD*C		**	**	**	**	**	**	**	**	**
Overall		1.88±0.008	2.87±0.012	0.87±0.004	0.41±0.003	0.69±0.006	0.91±0.010	2.85±0.024	1.25±0.012	0.45±0.003

Table 7. Least squares means and standard errors for investigated characteristics belonging to 2023.

Factors	N	CYS	VAL	MET	PHE	ILE	LEU	LYS	PRO
Sowing Date	N	P=0.911	P=0.003	P=0.000	P=0.349	P=0.308	P=0.000	P=0.255	P=0.063
1(November 15^th)	15	2.24±0.034	0.99±0.010^a	0.16±0.005^a	0.75±0.009	0.79±0.008	1.30±0.010^b	0.81±0.009	0.71±0.016
2(November 30^th)	15	2.24±0.034	0.94±0.010^b	0.13±0.005^b	0.73±0.009	0.77±0.008	1.25±0.010^c	0.79±0.009	0.65±0.016
3(December 15^th)	15	2.26±0.034	0.96±0.010^b	0.13±0.005^b	0.74±0.009	0.78±0.008	1.36±0.010^a	0.80±0.009	0.68±0.016
Cultivars		P=0.109	P=0.001	P=0.194	P=0.000	P=0.000	P=0.000	P=0.002	P=0.005
Deren	9	2.28±0.044^ab	0.94±0.013^a	0.14±0.007	0.73±0.012^ab	0.79±0.01^ab	1.29±0.012^b	0.78±0.011^bc	0.64±0.021^b
Irmak	9	2.25±0.044^ab	0.95±0.013^ab	0.13±0.007	0.71±0.012^c	0.78±0.01^c	1.30±0.012^b	0.80±0.011^ab	0.75±0.021^a
Karina	9	2.14±0.044^b	1.00±0.013^c	0.14±0.007	0.79±0.012^a	0.78±0.01^b	1.28±0.012^b	0.77±0.011^c	0.70±0.021^ab
Misya	9	2.23±0.044^ab	0.93±0.013^a	0.13±0.007	0.71±0.012^c	0.73±0.01^c	1.36±0.012^a	0.81±0.011^a	0.66±0.021^b
Local	9	2.31±0.044^b	0.98±0.013^bc	0.15±0.007	0.75±0.012^b	0.81±0.01^c	1.29±0.012^b	0.83±0.011^a	0.65±0.021^b
SD*C		**	**	**	**	**	**	**	**
Overall		2.24±0.020	0.96±0.006	0.14±0.003	0.74±0.005	0.78±0.004	1.31±0.006	0.8±0.005	0.68±0.009

The effect of sowing date and cultivar factors on arginine, alanine, triazine and cysteine, which are non-essential amino acids, was found to be statistically significant. In terms of non-essential amino acid content, peas rank 2nd after lentils [57]. In terms of arginine, sowing times were ranked as 3(2.88g/100 g)>2(2.84g/100 g)>1(2.82g/100 g) (Table 6). According to the varieties, the mean values were given as Misya (2.93g/100 g)>Deren 2.89 g/100g)>Karina (2.87g/100 g)>Irmak (2.84g/100 g)>Local (2.70g/100 g) (Table 6). The effect of sowing date and cultivars on alanine was found statistically significant. Sowing dates were ranked as 2 (1.35g/100 g)>1 (1.29g/100 g)>3 (1.28g/100 g) (Table 4). Average values of varieties were ranked as Karina (1.35g/100 g)>Deren =Misya (1.33g/100 g)>Irmak (1.29g/100 g)>Local (1.22 g/100 g) (Table 4). The effect of sowing date and cultivars on triazine was found to be statistically significant. Sowing dates are ordered as 2(0.53 g/100 g)>3=1 (0.52 g/100 g) (Table 4). The mean values of the varieties were as follows: Misya (0.56 g/100 g)>Irmak (0.54 g/100 g)>Local (0.52 g/100 g)>Karina (0.50 g/100 g)>Deren (0.49 g/100 g) (Table 4). The effect of sowing date and cultivars on cysteine was found statistically significant. Cysteine values varied between 2.14- 2.32 g/100 g (Table 5 and Table 7). The highest mean cysteine value (2.27 g/100 g) was measured at the third sowing date. The mean values of the varieties were Local (2.32 g/100 g)>Deren (2.29 g/100 g)>Irmak (2.26 g/100 g)>Misya (2.24 g/100 g)>Karina (2.16 g/100 g) (Table 5). In previous studies, arginine (1.93 g/100g), cysteine (0.31 g/100g), alanine (1.09 g/100g) were found to be lower in peas, while tyrosine (0.73 g/100g) was higher [58]. However, in this study, the amount of cysteine was found to be higher than in previous studies, so the result is remarkable in this respect. The difference in cultivars planted may have caused this result because, amino acid profiles of proteins in leguminous seeds are unbalanced [53]. They also stated that heritability estimates for percentage protein range from 0.25 to 0.60, according to species, genotypes within species, and environments. The decreased dry grain yield on the first planting date may have caused the high protein content. This decrease in protein ratio may affect amino acid composition. For this reason, the amounts of some amino acids may have increased at the time of first sowing. It is possible to encounter the same results in similar studies [51,59].

Early sowing is generally preferred for pea plants to ensure that the flowering period is completed before the cold period sets in. However, in recent years, the dry and warm period continues from late November to December, with lower night temperatures. In particular, plants planted at the first sowing date were exposed to low temperatures in January and took a long time to recover from the damage. The spring season has shortened and maximum temperatures of between 35˚C and 40˚C have been observed in late April and early May. These high temperature levels may cause an increase in saponin levels in plants. Saponins are antinutritional compounds that increase in plants under extreme conditions [60]. In the present study, saponin values were seen to increase with delayed sowing date. The order of the highest to lowest saponin content between sowing times is as follows 3>1>2 (Table 2 and Table 3). The highest mean saponin values were obtained at the third sowing date, especially in the Misya, Deren and Local varieties (96.74 mg/g, 65.47 mg/g and 64.16 mg/g, Table 2, respectively). The findings of this study are consistent with others. Studies have shown that winter varieties of pulses can contain more defensins [61]. Spring varieties show a greater response to cold induction and acclimation than fall varieties. It may be that, in 2021-22, the disease pressure was substantially greater than 2022-23. More precipitation during flowering and pod formation (April and May) is the most susceptible vegetation period [62].

Prolonged droughts and high temperatures tend to delays in sowing dates in regions like in our study, where the Mediterranean climate prevails and peas are not irrigated. Sowing can only be carried out after the expected rains in late November and December. For this reason, plants may be damaged by sudden cold snaps in January and February. All the varieties were affected by cold. The fact that Karina is a faster growing variety than the others caused it to be damaged by cold. However, it performed well in terms of tolerating cold damage. Misya was also affected by the cold, but high yields were obtained thanks to the optimum temperatures that prevailed during the flowering and pod formation periods. Karina was the most affected by cold and early sowing with a score of 5 for the first and second sowing times. However, this score decreased to 2 for the third sowing date. Deren (5) and Irmak (4) were also negatively affected by cold. Local (2) was the variety least affected by extreme climatic conditions.

Figure 2. Boxplots comparing results obtained for yield and cold damage (score:0-5) in peas. (x axis; seed yield (fresh&dry) and cold damage; y axis; Distribution of data from the experiment. Red:first sowing date Blue:second sowing date; Yellow:third sowing date) t ha^-1.

Figure 3. Boxplots comparing results obtained for saponin, phenolic and protein content in peas. (X axis; saponin (mg/g), phenolic; mg (GAE)/100g; protein (%); y axis; Distribution of data from the experiment. Red: first sowing date Blue: second sowing date; Yellow: third sowing date).

Figure 4. Box plots comparing the results obtained for amino acid content in peas. (X axis; Measured all amino acids (g 100 g^-1); y axis; Distribution of data from the experiment. Red: first sowing date blue: second sowing date; yellow: third sowing date). ASP: Aspartic acid, GLU: Glutamic acid, SER: Serine, HIS: Histidine, GLY: Glycine, THR: Threonine, ARG: Arginine, ALA: Alanine, TYR: Tyrosine, CYS: Cysteine, VAL: Valine, MET: Methionine, PHE: Phenylalanine, ILE: Isoleucine, LYS: Lysine, LEU: Leucine, PRO: Proline.

4. Conclusion

In this study on the pea, differences in sowing dates and cultivars were found to have significant effects on fresh and dry seed yield, saponin and total total phenolic content, and the levels of protein and amino acid composition. Sowing on November 30^th (the second sowing date) led to the highest fresh seed yield value (3.27 t ha^-1) (Table 3). According to cultivars, the highest fresh yield values were obtained from Misya (2.88 t ha^-1) (Table 2). According to the results, the varieties gave different responses at the first and second sowing date but the highest interaction for fresh yield was in Misya on 30 November sowing date. In terms of seed dry yields, the highest yield (1.85 t ha^-1) was at the third sowing date (Table 3). Among the varieties, Local has the highest dry seed yield (1.76 t ha^-1) (Table 3).^. In dry seed yield, Local variety had higher yield than the others (Table 3). The interaction for the highest dry seed yield was Local (Table 3) at the 15 December sowing date. It is evident that with the changing climatic conditions, it would be beneficial to have the flexibility to plant varieties such as the local ones, which are common in the region, at a later date than the traditional sowing date.

Saponin values were also significantly affected by the sowing date, with the highest saponin values obtained from samples planted on December 15^th. Peas planted at the third sowing date reached the pod formation stage in March and the high temperatures observed in this month caused an increase in their saponin content. This increase varied depending on the cultivar: the highest saponin value (96.79 mg/g) was obtained from Misya (Table 3). Phenolic compounds were also affected by temperatures and the highest phenolic compound content was again obtained from samples sown on December 15^th. Again, the difference between the varieties was significant and the highest value was obtained from Misya. The similarity of the results in terms of both saponin and phenolic contents may be a result of the cold damage that the plants encountered in the third period. In terms of protein content as well, sowing dates were found to have a significant effect. The highest protein content (27.75%) (Table 2) was obtained from samples planted on November 15^th (the first sowing date). Among the varieties Misya (28.77 %) and Deren (28.48%) had the highest protein content (Table 2). Seed protein content, amino acid levels and composition varied depending on the sowing dates. The response of different varieties to protein content varied depending on the sowing dates. Deren and Misya produced better results at all sowing dates. The other varieties exhibited the highest protein content on the first sowing date. Different sowing dates may result in different levels of protein content due to the temperatures encountered at the seed filling stage. Sowing dates also had a significant effect on amino acids. However, the performance of the varieties may vary according to the sowing date.

According to the study results, valine, methionine, phenylalanine, isoleucine, lysine, threonine and histidine values were found to be highest at the first sowing date (Figure 4). In terms of these amino acids, Local, Karina and Irmak exhibited higher values than the others at the first sowing date. However, Misya and Deren showed higher average values at the third sowing date. The amino acid composition was positively and significantly affected by early sowing (November 15^th). According to the results obtained in general, early sowing may have led to longer periods of seed filling and nitrogen accumulation in the seed, thus resulting in a high protein ratio.

The amino acid composition was affected by environmental factors during the seed filling and pod formation periods at different sowing dates. Average monthly air temperature, long-term mean temperature, and cumulative solar radiation during seed filling, as well as precipitation throughout the entire reproductive period, along with combinations of these climatic variables, might be significant explanatory factors for all amino acids. Each amino acid could exhibit different behavior depending on environmental conditions, suggesting compensatory effects among them.

The impact of sowing date on yield, protein content, and essential amino acids such as lysine, methionine, and histidine was determined. Considering this effect under changing climatic conditions could be beneficial for feeding the world's population.

Data Availability Statement

The data analyzed or generated during this study are publicly accessible at “The Effect of Sowing Date and Cultivars on Yield and Quality of Pea” under the reference number “agriculture-2776695”. No new data set was created in our study and/or data cannot be publicly shared due to confidentiality reasons.”.

Acknowledgments

This study received financial support from the Adnan Menderes University scientific research projects institution as a research project (ZRF-21043).

Conflicts of Interest

The author declares no conflict of interest.

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Figure 1. Climatic characteristics of the experimental area. (temp: average temperature(⁰C); Long term:long term average temperature(⁰C); pre: precipitation (mm); Long pre: long term precipitation(mm)).

Table 1. Soil properties of the experimental area.

Soil Texture			pH	Organic Matter	Phosphorus	Calcium	Sodium (ppm)
Sand (%)	Silt (%)	Clay (%)		(%)	(ppm)	(ppm)	Sodium (ppm)
72	16.7	11.3	8	1.2	21	2978	101
Sandy loam			High	Low	High	Low	Low

Table 2. Least squares means and standard errors for investigated characteristics belonging to 2022.

Factors	N	Yield (Fresh seed) (t ha^-1)	Yield (Dry seed) (t ha^-1)	Saponin (mg g^-1)	Phenolic mg (GAE)/100g	Protein (%)
Sowing Date	N	P=0.000	P=0.000	P=0.000	P=0.000	P=0.005
1(November 15^th)	15	2.18±0.081^b	0.87±0.032^c	57.95±4.007^a	35.82±9.211^b	27.75±0.265^a
2(November 30^th)	15	3.22±0.081^a	1.29±0.032^b	49.76±4.007^a	33.30±9.211^b	26.55±0.265^b
3(December 15^th)	15	2.83±0.081^b	1.40±0.032^a	102.35±4.007^b	127.30±9.211^a	26.67±0.265^b
Cultivars		P=0.117	P=0.752	P=0.000	P=0.002	P=0.000
Deren	9	2.79±0.104	1.19±0.041	65.47±5.173^a	53.63±11.891^b	28.48±0.342^a
Irmak	9	2.73±0.104	1.21±0.041	59.03±5.173^a	47.78±11.891^b	25.88±0.342^bc
Karina	9	2.50±0.104	1.14±0.041	64.68±5.173^a	38.20±11.891^b	26.67±0.342^c
Misya	9	2.88±0.104	1.19±0.041	96.74±5.173^b	98.72±11.891^a	28.77±0.342^a
Local	9	2.81±0.104	1.20±0.041	64.16±5.173^a	89.03±11.891^a	25.16±0.342^b
SD*C		**	**	**	**	**
Overall		2.74±0.047	1.19±0.018	70.02±2.313	65.47±5.318	26.99±0.153

SD*C : Interaction between sowing date x Cultivars **:P<0.01.

Table 3. Least squares means and standard errors for investigated characteristics belonging to 2023.

Factors	N	Yield (Freshseed) (t ha^-1)	Yield (Dryseed) (t ha^-1)	Saponin (mg g-1)	Phenolic mg (GAE)/100g	Protein (%)
Sowing Date	N	P=0.000	P=0.000	P=0.000	P=0.000	P=0.000
1(November 15^th)	15	2.10±0.081^c	1.55±0.002^a	58.01±4.007^a	35.71±9.211^a	27.12±0.260^a
2(November 30^th)	15	3.27±0.081^a	1.70±0.002^c	49.81±4.007^a	33.19±9.211^a	25.68±0.260^b
3(December 15^th)	15	2.78±0.081^b	1.85±0.002b	102.40±4.007^b	127.19±9.211^b	24.88±0.260^c
Cultivars		P=0.117	P=0.000	P=0.000	P=0.002	P=0.000
Deren	9	2.77±0.104	1.64±0.003^e	65.53±5.173^a	53.52±11.891^b	27.50±0.336^a
Irmak	9	2.70±0.104	1.70±0.003^c	59.09±5.173^a	47.67±11.891^b	25.45±0.336^b
Karina	9	2.47±0.104	1.73±0.003^b	64.74±5.173^a	38.09±11.891^b	25.91±0.336^bc
Misya	9	2.85±0.104	1.67±0.003^d	96.79±5.173^b	98.61±11.891^a	26.55±0.336^c
Local	9	2.79±0.104	1.76±0.003^a	64.22±5.173^a	88.92±11.891^a	24.06±0.336^d
SD*C		**	**	**	**	**
Overall		2.72±0.047	1.7±0.001	70.07±2.314	65.36±5.318	25.89±0.150

SD*C : Interaction between sowing date x Cultivars **:P<0.01.

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Submitted:

22 January 2024

Posted:

23 January 2024

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Subject: Environmental and Earth Sciences - Other

1. Introduction